Immunization of humans against enterotoxogenic infection by Escherichia coli

ABSTRACT

There is provided a vaccine material capable of providing substantial levels of protection against human disease caused by infection with Escherichia coli. The protecting means comprises pili of the infecting organism. The protection is given either by administering the pili directly to the subject to be protected or to a pregnant or lactating female where protection of the newborn is desired.

RELATED APPLICATIONS

This application is a continuation-in-part of applicants copendingapplication Ser. No. 854,343 filed Nov. 23, 1977, now U.S. Pat. No.4,237,115 issued Dec. 2, 1980.

BACKGROUND OF THE INVENTION

Escherichia coli is a ubiquitous pathogen of man and animals causing awide variety of diseases of clinical and economic importance. Thegreatest cause of mortality in the world is infant death and E. colidiarrheal disease in areas of poor sanitation is one of the mostimportant causes. E. coli has been estimated to cause about 40% of alltravelers diarrhea. E. coli is the most frequent cause ofhospital-acquired infections and infections of debilitated andtraumatized persons. Bacteremias and burn-wound infections arefrequently due to E. coli. E. coli is the most frequent cause of urinarytract infections causing acute and chronic cystitis and pyelonephritis.It is clear that the development of methods to prevent E. coli diseasewould alleviate much human misery and stop huge economic losses.

Applicant has surveyed a large number of pathogenic strains of E. colifrom a number of human diseases and established that the great majorityof them possess pili belonging to a single immunologically relatedfamily named "E. coli Type 1" pili. In a co-pending application (BTX3.0-005) U.S. Ser. No. 187,049 filed Sept. 15, 1980 which isincorporated herewith by reference, there is disclosed a generalmethodology of serological analysis. This methodology shows that membersof the E. coli Type 1 pilus family can be ranked in a hierarchial orderfrom most senior to most junior. Antisera to the most senior pilus typecan cross-react to a large extent with all other members in the family.Therefore, provision of a vaccine consisting of a single type ofpurified E. coli Type 1 pili would protect against the great majority ofE. coli infections of humans, that is, infections caused by strainsbearing pili in the Type 1 family especially those strains with pilijunior to the pili in the vaccine. There also exist otherimmunologically related families of pili on Escherichia coli strainspathogenic for humans. These families are unrelated immunologically toType 1 pili associated with mannose-sensitive hemagglutination of guineapig erythocytes. These are known as NSM pili. Vaccines derived from NMSpili would also be useful in providing protection against the various E.coli infections.

SUMMARY OF THE INVENTION

There are provided vaccine compositions capable of raising the antibodylevel of a human subject to a level sufficient to provide protectionagainst infection caused by organisms of a first group of strains ofpiliated E. coli comprising:

(a) Pili derived from a second group of strains of piliated E. coliorganisms wherein cells of organisms of said first group arecross-reactive with serum containing antibodies against pili from saidsecond group, said first group consisting of strains which may be thesame or different from, and suitably junior to, the strains of saidsecond group; and,

(b) a pharmaceutically acceptable carrier.

There is exemplified in this application a vaccine containing pili froma single predetermined strain which is effective against infectioncaused by organisms of the same strain. Vaccines which contain both Type1 and NMS pili are also included within the scope of the presentinvention provided, of course, that of said pili will cross-reactimmunologically with at least one of the infecting organisms.

An important enterotoxogenic strain of E. coli was isolated from humanswith diarrhea and shown to be virulent and causative of the naturaldisease when inoculated into non-immune humans. This virulent strain,though sparsely piliated, was grown and selected for well-piliatedclones. These clones were grown and maintained on minimum glucose baseagar medium and the pili therefrom separated from the cells andsubjected to several cycles of crystallization in aqueous magnesiumchloride followed by resolubilization in low ionic strength neutralbuffer.

Test subjects were injected with pili. Thereafter, the subjects werechallenged intragastrically with an amount of E. coli previously foundto cause infection in non-immune subjects.

Although incidence of diarrhea was noted in the immunized group, membersof this group recovered rapidly relative to the non-immunized challengegroup.

Culture of E. coli H 10407

Samples of a parental strain E. coli H 10407 (078:H11) was isolated froma patient suffering from severe diarrhea in Bangladesh. The sample waspassed through still broth, and colonial forms selected therefrom toprovide a well piliated clone designated E. coli H 10407 (ATCC 31703).The clone is then grown on minimal glucose agar base medium. The piliwere separated from the cells by blending and centrifugation in a lowionic strength neutral buffer such as phosphate buffer and saline pH7.0. The pili are crystallized from the buffer by addition thereto ofconcentrated magnesium chloride (aq) to bring the strength of the bufferup to the 0.10 M whereupon the pili crystallize. The crystalline piliare taken up in a low ionic strength neutral buffer such as 0.04 Mphosphate buffer pH 7.0 (without saline) and reprecipitated withmagnesium chloride in a similar manner. It is preferred to subject thepili to from one to five cycles of crystallization. The procedure usedis that substantially set forth in Brinton, Trans. N.Y. Acad. Sci 27,1003 (1965).

The final preparation of the pilus vaccine consists of blending andfiltering the recrystallized pili through sterile filters suitably witha preservative such as merthiolate. The pili thus prepared are of aquality sufficient to pass the standards of the Burean of Biologics,Food and Drug Administration for general safety, sterility, andpyrogenicity.

The pili may be administered orally--say, in capsule form--or byinjection--that is to say, subcutaneous, intradermal, or intramuscularinjections. Where the mode of administration is by injection, since thepili are solid, any pharmaceutically acceptable suspending medium may beemployed. It has been found especially useful to employ phosphatebuffer, suitably containing merthiolate, as the vehicle or suspendingmedium. It is preferred to use 0.005-0.1, most suitably 0.04, ionicstrength phosphate buffer containing 0.005 to 0.1, most suitably 0.01%,merthiolate. The concentration of NMS pili (associated withnon-mannose-sensitive hemagglutination of human erythocytes in thevehicle is not critical. The sole criterion of desirability being thatthe pili shall be sufficiently finely divided to provide a suspensionwhich meets generally accepted standards of syringeability. Aconcentration of 1-30, preferably about 20 mg of pilus protein per 10 mlof suspending medium is especially suitable.

It is generally preferred to administer the vaccine composition in morethan one dose separated by a predetermined time factor. This time factoris selected to permit the formation of an adequate titer of antibodiesto the pili in the injected subject. It has been found suitable toadminister the vaccine composition at least once, preferably at 60 andagain at 30 days pre-infection.

Since there are no local or systemic toxic effects engendered by theinjection of vaccine, there appear to be no upper limits to the dosageadministered. It has been found suitable, however, to administer between1 and 100 micrograms of pili per kilogram of body weight, most suitablyabout 20 milograms per kilogram of body weight per injection.

E. coli infection occurs, due to many different strains thereof, indifferent species of mammals. It has been found that E. coli specieshaving Type 1 pili are responsible for human infections. Thus, the piliderived from related members of said group of species will provideprotecting antibodies in a system into which they are administered.

EXAMPLE I Preparation of Escherichia Coli Pili (Type)

A culture prepared by resuspending piliated phase colonies growing onminimal glucose agar base medium in a liquid 2 broth (1% Bactotryptone,0.8% NaCl, 0.1% yeast extract, 0.1% glucose) medium was used toinoculate trays containing the same growth medium solidified with agar.After overnight incubation at 37° C., the confluent bacterial growth wassuspended in 0.01 molar phosphate buffered saline pH 7.0. About twentymilliliters of buffer was used to suspend the growth from one tray whichdimensions were approximately 30 cm×40 cm. The resuspended growth wasblended, 200 milliliters at a time, at 14,000 rpm for five minutes inthe 400 milliliter cup of a Sorvall OMNIMIXER in order to remove pilifrom the cells. Cells were then removed by centrifugation at 10,000times G for twenty minutes and the supernatant liquid was retained. Thepili were then crystallized by the addition of magnesium chloride(MgCl₂) to 0.1 molar. After the crystals formed, they were removed fromsuspension by centrifugation at 20,000 times G for sixty minutes, andthe pellet was retained. The pellet containing the pilus crystals wasredissolved in 0.04 phosphate molar buffer pH 7.02 (without saline). Thesuspension was clarified by centrifugation at 20,000 times G for sixtyminutes and the supernatant liquid was retained. The cycle ofcrystallization, centrifugation, redissolution, and centrifugation wasrepeated two to four times to obtain the purified pilus suspension.

EXAMPLE II Vaccine Preparation

(a) High speed blending with Sorvall Omnimix at 14,000 rpm for 10minutes with 100 ml aliquots in a 200 ml cup, broke up pilus aggregatesand shears the rods 0.01% merthiolate added and

(b) Concentration was determined by U.V and adjusted to a 1800 mcg/10 mldosage form.

Removal of Flagella

Cycled solubilized pili preparation were adjusted to pH 12-3 by additionof 1 N sodium hydroxide, after 30 minutes, at room temperature withoccasional stirring, the pili were precipitated by addition of 10% (u/v)saturated ammonium sulphate followed by centrifugation at 20,000 G for30 minutes leaving dissociated flagella subunits in the supernate.

The precipitated pili were then resolubilized in phosphate buffer (0.04M).

EXAMPLE III Immunization

Sequential groups of 3-6 volunteers (21 in total) were parenterallyinoculated in the triceps muscle with 45, 90, 90 or 1800 mcg doses ofpurified pili vaccine. Twenty-eight days thereafter 15 of the 21volunteers received a booster IM inoculation with 1800 mcg of pilivaccine. Volunteers who received 1800 mcg primary or boosterinoculations were also given an order to assess local reactions due topili rather than the act of IM innoculation per se. Neither thevolunteer nor the examining physician was told which ar received vaccineand which received saline.

Challenge Study

Approximately one month following inoculation with the 18 mcg boosterdose (two months after primary immunization), six vaccines and sevenunimmunized control volunteers participated in a challenge study toassess vaccine efficacy.

EXAMPLE IV Preparation of Inocula

The E. coli H 10407 parent strain utilized in previous challenge studieswas inoculated into Z broth, incubated for 4 hours with shaking at 30°C. and frozen at -70° C. with DMSO. The frozen stock was later thawedand streaked onto casamino-yeast extract (CAYE) agar plates.

Inocula and Challenge

After 15 hours incubation at 30° C. 16 piliated colonies identifiedunder stereo-microscope were picked and streaked on CAYE agar. Twelvehours after incubation at 37° C., 30 piliated colonies were used toheaving inoculate each of six CAYE agar plates for incubation at 37° C.After twelve hours, the CAYE agar cultures were harvested with saline(0.85%) and dilutions made in saline.

EXAMPLE V Administration of Inoculum

Two gm of NaHCO₃ were dissolved in 150 ml of distilled water of whichthe volunteers drank 120 ml; one minute later they drank the remaining30 ml in which the E. coli inoculum (5×10⁸ organisms) was suspended.Inoculum size was quantitated by replicate pour-plate technique beforeand after challenge. The presence of both type 1 somatic and NMSpiliation on the challenge organisms was documented by agglutinationwith specific antisera.

Clinical Observations Immunization

Volunteers were kept under close observation on the Isolation Ward fortwo days post-inoculation with pili vaccine. Oral temperatures weretaken every six hours and injection sites were examined for erythema,heat, induration and tenderness.

Challenge

Volunteers were examined daily starting three days prior to ingestingthe virulent organisms. Oral temperatures were taken every six hours andrepeated within five minutes if they were 37° C. or above. All stoolsand vomitus were collected in plastic cholera seats, examined by a nurseor physician and volumes measured. Stools were graded on a five pointscale--grades 1 (fully formed) and 2 (soft) were considered normal;grade 3 denoted thick liquid stool, grade 4 opaque-watery, and grade 5rice-water stools. Diarrhea was defined as three or more loose (grade3-5) stools in 24 hours or at least two loose stools within 24 hourssurpassing 250 ml in volume. Prior to discharge all volunteers receiveda five-day course of oral neomycin (500 mg six hourly) to eradicatefecal carriage of the virulent ETEC strain.

RESULTS Reactogenicity

Clinical:

Neither erythema, induration, heat, tenderness, fever nor malaiseoccurred in any of the 21 volunteers who received primary immunizationwith varying dose of purified pili vaccine (Table 1). Among the fifteenpersons who received an 1800 mcg booster inoculation no systemic adversereactions were noted but six vaccinees developed objective local adversereactions including induration, heat or erythema (Table 2). Localreaction following the booster occurred in persons who had receivedprimary inoculations with 45 (2), 900 (2) and 1800 (2) mcg doses ofvaccine. Local reactions were evident 24 hours post-inoculation but,with one exception, were gone by 48 hours. The reactions were describedas mild to moderate by the volunteers. In no instance did nausea,vomiting, diarrhea or fever occur.

Vaccine Efficacy

Clinical:

One month after IM inoculation with an 1800 mcg booster dose of pilivaccine, six vaccinees agreed to participate in a challenge study alongwith seven unimmunized control volunteers. Following ingestion of 5×10⁸virulent E. coli H 10407 bacteria all seven controls developed diarrhealillness (Table 5). Three controls passed copious rice-water stoolsresulting in chloera-like total diarrheal stool volumes of 3.8, 7.5 and9.9 liters; two controls required intravenous fluids to maintainhydration. In contrast, only 2 of 6 vaccinees developed diarrhealillness (p=0.04, Fisher's Exact Test). While ill controls experiencedmalaise (7 of 7) and vomiting (6 of 7), none of the vaccinees, ill orwell, had these complaints (Table 5). Otherwise, the diarrheal illnessmanifested by the two vaccinees was similar in incubation, total volumenumber of loose stools and duration to that seen in the controls.

                                      TABLE 1                                     __________________________________________________________________________    RESPONSE OF VACCINEES IMMUNIZED WITH TWO PARENTERAL                           DOSES OF PURIFIED E. COLI TYPE 1 SOMATIC PILI VACCINE AND CONTROLS            FOLLOWING INGESTION OF 5 × 10.sup.8 VIRULENT ENTEROTOXIGENIC E.         COLI (STRAIN H10407)                                                                                Mean Total Diarrheal                                                                      Mean Total No.          Positive            Mean                  Stool Volume per                                                                          Loose Stools per        Stool               Incubation (hrs)                                                                           Diarrhea Ill Volunteer                                                                             Ill Volunteer                                                                          Vomiting                                                                            Malaise                                                                            Fever                                                                             Culture             __________________________________________________________________________    Vaccinees                                                                          36.5                                                                                   ##STR1##                                                                              3.89.sup.+ (1.38-6.40)**                                                                  16 (15-19)**                                                                           0/6   0/6  0/6 6/6                 Controls                                                                           34.5             3.96        18       6/7   7/7  2/7 7/7                                       (1.37-9.86) (7-29)                                      __________________________________________________________________________     *No. positive/No. challenged                                                  .sup.+ liters                                                                 **(range)                                                                

                                      TABLE 2                                     __________________________________________________________________________    CLINICAL RESPONSE OF VOLUNTEERS TO PARENTERAL                                 IMMUNIZATION WITH PURIFIED H10407 TYPE 1 SOMATIC                              PILI VACCINE                                                                  Following Initial Vaccine Dose                                                                       Following 1800 mcg. Booster Dose                       Dose  Fever                                                                             Malaise                                                                            Local Reactions                                                                       Fever                                                                             Malaise                                                                            Local Reactions                               __________________________________________________________________________     45 mcgs.                                                                            0/3*                                                                             0/3  0/3     0/3 0/3  2/3                                            90 mcgs.                                                                           0/4 0/4  0/4     0/3 0/3  0/3                                            900 mcgs.                                                                          0/4 0/4  0/4     0/3 0/3  2/3                                           1800 mcgs.                                                                           0/10                                                                              0/10                                                                               0/10   0/6 0/6  2/6                                           __________________________________________________________________________     *No. with reactions/No. immunized                                        

EXAMPLE VII

In my co-pending application filed contemporaneously herewith (BTX3.0-005) U.S. Ser. No. 187,049, there is disclosed the concept ofhierarchy of strains within a group of immunologically related piliatedorganisms. In said application I show how the senior organisms of thegroup may be identified and said senior organisms protect againstinfection by junior organisms. The disclosure of said application isincorporated herein by reference.

Hence with respect to E. coli vaccines, compositions derived from seniorstrains will protect against infections by junior strains.

In Table 3 below there is shown a normalized cross-reactivity tableunder the principles of my co-pending application therefore vaccinesproduced as shown from E25 (ATCC 31703) pili will protect againstinfection by strains junior thereto including H 10407 (ATCC 31705).

While these principles are disclosed with respect to type 1 pili, theyare equally applicable to the NMS pili, vaccines derived from which areexpressly included within the scope of the present invention.

                  TABLE 3                                                         ______________________________________                                        TABLE OF SENIOR/JUNIOR HIERARCHY                                              E. COLI TYPE 1 PILUS FAMILY                                                          Antisera                                                                Pili    E25      B9     H10407   TD   E28                                    ______________________________________                                        E25      100      62      8       10    2                                     B9       142      100     9       14    2                                     H10407    16      25     100       8   14                                     TD        48      57     31       100  12                                     E28       34      41     51       19   100                                    ______________________________________                                    

EXAMPLE VIII

(a) Isolation and characterization of piliated phase E. coli (NMS Pili)

The original H 10407 parent culture was passed on casamino acid-yeastextract (CAYE) agar (Appendix), and resulting colonies were screened forhemagglutination of human red blood cells that was not sensitive toinhibition with D-mannose (NMS=non-mannose sensitive). When such a clonewas detected, it was passed continuously on CAYE agar. Piliation wasverified by electron microscopic examination and was observed to bemorphologically distinguishable from Type 1 piliated clones of H 10407isolated from the same parent. NMS pili are rods 50-60 A in diameterlike Type 1 pili, but they are more flexible and unwind to form thinfibers (10-20 A diameter).

The NMS colony type is smaller and lighter in pigmentation than the Type1 piliated or nonpiliated colony types when grown under theseconditions. When NMS clones are passed continuously on CAYE plates thereare very few reversions detectable after 10-14 hours at 37° C.

H 10407 can produce two different kinds of hemagglutinins. One kind,Type 1 pili, participates in a mannose-sensitive hemagglutination withguinea pig and human red blood cells and has been shown to be Type 1pili. The other kind of hemagglutin is responsible for anon-mannose-sensitive hemagglutination with human red blood cells butnot guinea pig cells. Clones possessing the latter factor are referredto as NMSP+.

(b) Growth

Because the subtle differences in NMS colonial morphology could best bedetected in 10-14 hour cultures, clones were maintained by continuoussubculture every 12 hours on CAYE agar. Five to six colonies were pickedto avoid selecting atypical mutants. Furthermore, growth in liquid mediaseverely depleted the cultures of NMS-piliated cells. Therefore,cultures for the inoculation of large growth trays (101/2×151/2×1 inch)had to be prepared on solid media both using CAYE agar. Growth forinoculation was harvested off CAYE agar in petri dishes using 7.5 ml of0.7% casamino acids for dish and scraping the agar surface with a bentglass rod. The suspended growth was then pipetted out of the dish andused to inoculate 2 large growth trays, 2-3 ml per tray. Afterinoculation, the trays were covered with tightly fitted aluminum lidsand incubated for 19-21 hours at 37° and 70% relative humidity in adedicated floor model humidified incubator located in the adjacent room.

After incubation, the growth trays were returned to the vaccine facilityfor harvesting. Trays were inspected visually for contamination.Cultures were harvested with the addition of 5-8 ml harvest buffer usinga clean glass plate to scrape the growth off the agar surface. Suspendedgrowth was aspirated into a sterile flask and kept on ice until theentire day's harvest was collected. The polled harvest was then treatedas described in the following section.

(c) Isolation of NMS pili

To remove pili from the cells, the harvest suspension was blended in 200ml portions in an ice chilled cup using the Sorvall Omnimixer. Eachportion was blended for 5' at 11,700-13,000 rpm, as determined by 3 to 4readings with a tachometer. Approximately half of an 80 tray harvest wasblended and centrifuged before blending the remaining half. The blendedharvest was centrifuged at 15,380 g for 25 minutes to remove the cells.The cell pellets were discarded.

Supernatants of blended cultures were pooled and the volume measured.Crystalline ammonium sulfate was added slowly to 20% saturation withconstant stirring. (In contrast to Type 1 pili, NMS pili could not becrystallized by Mg++.) Streaming birefringence was immediately visible.The preparation was allowed to crystallize overnight in the cold. NMSpilus crystals were pelleted at 30,050 g for 60 minutes, and thesupernatant discarded. The pellets of crystalline pili were thensolubilized by magnetic stirring for about 1 hour, followed by standingin the cold overnight in solubilizing buffer. One liter of solubilizingbuffer was used per 80-tray harvest batch. The solubilized preparationwas clarified by centrifugation at 30,000 g for 60 minutes.

The cycle of crystallization/solubilization was repeated for a secondtime by measuring the exact volume of the clarified supernatant andadding crystalline ammonium sulfate to 20% saturation, slowly and withstiring. The preparation was allowed to sand in the cold overnight, thencentrifuged at 30,050 g to pellet the pilus crystals. The same sequenceas before was repeated for solubilization and clarification.

The third cycle of purification was the same as the second, except thatonly 500 ml of buffer were used to solubilize the crystals. Allpreparations were monitored by darkfield microscopy and SDS-PAGEthroughout the purification procedure.

Pilus preparations were allowed to equlibrate to room temperature forabout 20 minutes. The pH was then adjusted to 2.5 using 0.2 N HCl, withrapid stirring. The pH-adjusted solutions were allowed to stand at roomtemperature for 5-8 minutes, then immediatel centrifuged at 30,050 g for60 minutes. Pilus-containing supernatants were decanted and immediatelyreadjusted to pH 7.0-7.2 with 0,2 N NaOH. The brownish flagellarmaterial in the pellet was discarded. Crystalline ammonium sulfate wasadded to the supernatant to 20% saturation and crystallization wasallowed to continue overnight in the cold. At this point, preparationsat identical stages of purification were pooled, forming 2 pools of NMSpili for ease in handling. The flagella removal procedure was repeatedon these 2 pools until the amount of flagella was reduced to less than1%, as measured by SDS-PAGE.

EXAMPLE IIX Vaccine Preparation

The preparation was dilated to 0.5 mg/m/ and prefiltered once through a45μ Gelman filter using a 142 mm Millipore disc filtration unit with a 2liter capacity cylinder. Merthiolate is added to a final concentrationof 0.01% and the final vaccine material was then filter sterilized in asingle filling under positive pressure through the sterilized unit andcollected in a sterile receiving bottle.

Pilus vaccines have been shown to be optimally antigenic in rabbits atdoses of about 1 μg/kg body weight. For the average adult male of bodyweight 150 lbs, this requires injection of about 1.0 ml of vaccine at aconcentration of 0.5 mg/ml.

                  TABLE 3                                                         ______________________________________                                        ELISA Titers of Sera from NMS Immunized and Nonimmunized                      Humans                                                                        Sample        Standard Level                                                  ______________________________________                                        A        Day 18   100,000                                                     A        Preimm.  1,886                                                       A        Preimm.  2,349                                                       B                 1,600                                                       C                 1,742                                                       D                 1,839                                                       D                 1,769                                                       E                 1,481                                                       F                 1,781                                                       G                 2,145                                                       H                 2,600                                                       I                 1,397                                                       J                 1,390                                                       K                 1,798                                                       L                 1,539                                                       ______________________________________                                         ##STR2##   55-fold increase  x = 1,808                                                  In 18 days after 1 injection                                                                    = 352                                                       of 1.0 mg                                                                     6 μg/lb body weight                                             No systemic toxic effects. Only local soreness and redness.

EXAMPLE X Protection of Infants Against Coli bacillosis neonatorum

Pregnant mothers are inocculated with the E. Coli pilus vaccine as setforth above, suitably 60 and preferably again 30 days prepartum. Thenewborn infant is protected either by being suckled by the motherimmediately postpartum or the mother's colostrum is withdrawn and fed tothe infant. It should be noted that the antibodies are present in thecolostrum, thus protection may be provided to the newborn of others by aLactating immunized mother.

I claim:
 1. A method of protecting human subjects against colibacillosiscaused by a member of a predetermined first group of strains of piliatedE. coli which comprises administering to a subject in need of protectiona composition capable of raising the antibody level of a human subjectto a level sufficient to provide protection against infection caused bya member of a first group of strains of piliated E.coli comprisingpilipreviously separated from other E.coli organism components derived fromat least one member selected from a second group consisting of strainsof piliated E. coli organisms having Type 1 pili and those having NMSpili wherein cells of organisms of said first group are agglutinable byserum containing antibodies against pili from said second group, saidfirst group consisting of strains which may be the same as or differentfrom the strains of said group.
 2. A method of claim 1 wherein thevaccine composition is administered at least once to the subjectsbetween about 5 and about 60 days before infection.
 3. A method of claim2 wherein there is administered between 1 and 100 μg/Kg body weight ofthe pilic component of vaccine composition.
 4. A method of claim 1wherein the pilic component of the vaccine composition is derived fromType 1 pili or NMS pili of E. coli H 10407 (ATCC 31705) or E. coli E-25(ATCC 31703).